28 years after the beginning of the AIDS epidemic, we are confronting the fact that more than one out of three people with HIV-1 develop some form of HIV-1 associated neurocognitive disorder (HAND). Highly active antiretroviral therapy (HAART), even with dramatic reduction of viral load, may reverse some symptoms of HAND (often on a transient basis), but neither cures or prevents the disease. This RO1 has investigated the role of the phospholipid mediator platelet activating factor (PAF) in normal and pathologic synaptic transmission as a key molecule involved in the neuropathogenesis of HAND. In this competing renewal, we hypothesize that viral proteins from HIV-1 can disrupt normal PAF signaling between neuronal synapses by direct and enduring effects on CNS-trafficking immune effector cells that amplify the effects of PAF and cause neurologic disease. Building on data from the previous cycle of this RO1, prolonged exposure or increasing doses of PAF results in a continuum of reversible to irreversible synaptodendritic damage with failure of synaptic transmission due to mitochondrial dysfunction. In this application, we will use in vivo multiphoton microscopy (MPM) to investigate synaptodendritic damage during conditions that model HAND in mouse cortex, using transgenic animals with fluorescently labeled neurons and/or mononuclear phagocytes. We propose three specific aims using HIV-1 Tat and its downstream effector, PAF, to induce changes in immune cell trafficking into the CNS and synaptic communication. In aim 1, we will characterize in vivo PAF-induced dendritic beading that may become irreversible after PAF concentrations exceed thresholds for excitotoxic and neuroinflammatory damage, with the goal of understanding how increasing doses of PAF affect synaptic architecture and function during neuroinflammatory conditions that model HAND. In aim 2, we will use immunodepletion techniques to selectively remove peripheral mononuclear cells or neutrophils prior to stereotactic injection of HIV-1 Tat into the brain, to test whether these cells contribute to synaptodendritic damage by phagocytic engulfment or release of soluble mediators. In aim 3, we will determine the in vivo kinetics of cortical NADH production in response to Tat and PAF, as a measure of tissue oxygenation and energy metabolism; these experiments will test whether synaptodendritic damage occurs due to energy failure in response to increased synaptic activity during neuroinflammation. Together, these aims will help us translate our model of HIV-1 neuropathogenesis to in vivo conditions, bringing us closer to an understanding of how HAND continues to be a problem of major importance to people living with HIV-1.